The Toll-like receptors (TLRs) mediate innate immune recognition of pathogens in species as diverse as flies and humans. The purpose of this proposal is to analyze how the intracellular signaling domains of TLRs interact with each other to generate distinct pro-inflammatory signals in response to bacterial products. We demonstrated previously that while dimerization of the cytoplasmic tail of TLR4 is sufficient to induce the production of TNF-a, dimerization of the cytoplasmic tails of either TLR2 or TLR6 does not. In contrast, heterodimerization of the cytoplasmic tails of TLR2 and TLR6 does induce TNF-a. Thus, the mechanism of dimer-induced signaling is different between these two receptor pairs. We propose to map the elements of the cytoplasmic domain of TLR4 that mediate homodimer-induced signaling, and to map the elements of the cytoplasmic domains of TLR2/6 that mediate heterodimer-induced signaling. The biological consequences of these two types of signaling are different; while TLR4 homodimers induce the chemokine IP-lO, TLR2/6 heterodimers do not. We will define the regions of the cytoplasmic domain of TLR4 that specifically mediate the induction of IP- 10, and identify signaling molecules that bind to TLR4 homodimers, and not to TLR2/6 heterodimers. Although we have demonstrated previously that TLR4 mediates LPS-responses in macrophages, while TLR2/6 mediates responses to peptidoglycan, there is tantalizing evidence that under certain circumstances, TLR2 may participate in LPS-induced responses. One such circumstance is the induction of IL-12; one dominant negative mutant of TLR2 specifically ablates this response while a distinct TLR2 mutant does not. Interestingly, neither mutant blocks LPS-induced TNF-a. This distinction should permit us to map distinct areas of the cytoplasmic domain of TLR2 that participate in LPS-induced IL-12 production. We have used a rapid and robust method for mapping the signaling capacity of the cytoplasmic domains of TLR 2,4, and 6 dimers, and we will extend these studies to examine the capacity of the cytoplasmic domains of all ten TLRS to generate pro-inflammatory signals. This proposal will therefore define the signaling repertoire of different pairs of TLRs, as well as the regions of the cytoplasmic domains of these molecules responsible for triggering distinct responses such as those leading to TNF-cz, IP-lO, and IL-12.